A New Multilocus Sequence Typing Scheme for Lactobacillus delbrueckii subsp. bulgaricus Based on Whole-Genome Sequencing Data.

Abstract

In this study, we developed a novel 13 housekeeping gene multilocus sequence typing (MLST) scheme for Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) using whole-genome sequencing data, comprising 218 GenBank-retrieved and 29 newly isolated Turkish strains. We identified 291 distinct alleles across 13 housekeeping genes (clpX, pepX, pheS, mutL, murE, dnaA, gyrB, ddlA, pyrG, rpoB, fusA, recA, and tuf), with allele frequencies ranging from 2.7 to 10.9%, and defined 133 sequence types (STs), including 12 novel STs (ST122-ST133) unique to Turkish isolates, demonstrating superior discriminatory power compared to prior 8-gene MLST schemes (106 STs). This scheme offers a practical, standardized, and less computationally demanding alternative to core genome MLST (cgMLST) for large-scale epidemiological and industrial tracking, while maintaining high resolution. Population structure analysis revealed 17 clonal complexes (CC1-CC17) and 76 singleton STs (57.1%), with the largest complex (CC-1) containing 17 STs. Turkish isolates formed a distinct clonal complex (CC-6) or existed as singletons, underscoring their genetic uniqueness. Genetic diversity analyses showed significant differences between reference and Turkish populations, with nucleotide diversity (π) values of 0.00364 and 0.00225, respectively. The gyrB locus exhibited the highest diversity in reference strains (π = 0.00858), while pyrG and mutL showed the highest diversity in Turkish isolates. Significant linkage disequilibrium was detected in both populations ( $I_{\text{A}}^{\text{S}}$  = 0.2653 for reference strains; $I_{\text{A}}^{\text{S}}$  = 0.3395 for Turkish isolates), suggesting predominantly clonal population structures with limited recombination. Split decomposition and Phi test analyses confirmed the presence of recombination (P < 0.05) across the concatenated sequences. These findings enhance our understanding of L. bulgaricus population genetics and highlight the importance of geographical diversity in shaping bacterial evolution. This standardized MLST scheme provides a robust, scalable, and portable framework for starter culture selection and the tracking of traditional dairy microbiota, complementing higher-resolution whole-genome typing methods.